Escherichia coli O157:H7 is the most common infectious cause of bloody diarrhea, or hemorrhagic colitis (HC), in the U.S, and the incidence of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) is about half that of O157:H7. Moreover, the hemolytic uremic syndrome (HUS), a sequela of O157:H7 in particular but also other STEC infection, is the most frequent basis for acute kidney failure in U.S. children. E. coli O157:H7 belongs to a subset of STEC called Enterohemorrhagic E. coli (EHEC) that not only make Shiga toxin (Stx) type 1 or type 2 (or a variant thereof) but also harbor pO157 (or a related plasmid) and express the adhesin intimin, an outer membrane protein encoded by the eae gene contained within a pathogenicity island called the locus of enterocyte effacement. Intimin engages its bacterially-expressed translocated receptor Tir and also binds, albeit less avidly, to the eukaryotic receptor nucleolin. We recently found that Stx2 increased the level of nucleolin on the surface of HEp2 cells and, possibly as a consequence of such a toxin-mediated alteration of the cell surface of enterocytes, an Stx2-expressing EHEC O157 strain colonized the intestines of mice better than did its isogenic Stx2-negative mutant. The long-term goals of this project are to define at the molecular, cellular, and whole animal levels the pathogenic mechanisms by which STEC cause disease and to test strategies to prevent and treat these illnesses. The specific aims are to: 1. investigate whether both Stx1 and Stx2 contribute to the development of kidney lesions and/or other HUSrelated clinical features when produced from the same E. coli O157:H7 strain after oral inoculation of Dutch belted rabbits, a newly described model of HUS; 2. examine the basis for the enhanced virulence in orallyinfected, streptomycin-treated mice of STEC strains that produce the Stx2d-intestinal mucus activatable toxin (Stx2dact), an Stx2 variant that when produced by eae-negative STEC strains has been associated with bloody diarrhea and HUS in European patients; 3. continue to probe the role of Stx2 in the establishment of infection by E. coli O157:H7 in the murine gut, measure the impact of Stx2 on nucleolin expression in vivo, and test whether Stx1 can augment O157:H7 colonization; and, 4. determine whether Stx2 modulates nucleolin expression and/or distribution in epithelial cells and whether such changes are linked to Stx-induced apoptosis of intoxicated cells. Public health relevance: Due to the potential severity of O157:H7 infection and because the organism has a very low 50% infectious dose and can spread from person to person, E.coli O157:H7 is classified as a category B biological agent. As an outcome of this project, we will further delineate the steps by which this pathogen causes disease, which, in turn, may facilitate the creation of more targeted strategies to interfere with the infectious and intoxication processes.